Identification of a β-arrestin-biased negative allosteric modulator for the β₂-adrenergic receptor

Catecholamine-stimulated β₂-adrenergic receptor (β₂AR) signaling via the canonical G_s-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β₂AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β₂AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias β₂AR signaling toward the G_s pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β₂AR activation. Since attempts to develop G_s-biased agonists and allosteric modulators for the β₂AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit β-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of β-arrestin recruitment to the β₂AR while having no effect on β₂AR coupling to G_s. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the β₂AR and protects against the functional desensitization of β-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the β₂AR with minimal effects on the β₁AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the β₂AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the β₂AR.